Cloning, Expression And Characterization Of PnpC Gene From Nitrophenol Degradation Bacterium

Nitrophenols, the important raw materials, are widely used in the synthesis of pesticide, explosives, dye and other organic compounds. Some chemical products could be hydrolyzed to produce p-nitrophenol (PNP) in the environment, such as organophosphorus pesticide and nitrophenol herbicides. According to its biological toxicity and difficulty to degrade, PNP is defined as the top level of hazard pollutant by national standards. The PNP accumulation has become the attentive environment problem. Microbial enzyme hydrolysis of nitrophenol has developed as an important technology for nitrophenol remediation due to its advantages of high efficiency, low cost, and eco-friendly. In the present study, several pnpC genes encoding the key enzyme of hydroxyquinol-1,2-dioxygenase in PNP catabolism were cloned from bacteria with high ability to degrade PNP. The pnpC product (PnpC) was expressed and purified as recombinant proteins to investigate its expression and catalytic properties.With the genome DNA of Pseudomonas sp. HS-D38, Bacillus cereus HS-MP12 and Bacillus cereus HS-MP13 that could efficiently degrade PNP as templates, we amplified the hydroxyquinol-1,2-dioxygenase gene named pnpC1/2/3 by PCR. The PCR products were inserted into pMD18-T vector and then sub-cloned into the expression vector of pET-28. The recombinant plasmids of pET-pnpC1/2/3 were transformed into Escherichia coli BL21(DE3) for the Isopropylβ-D-1-Thiogalactopyranoside (IPTG) induced expression of target enzyme of PnpC1/2/3 at high level. In addition, we optimized the expression conditions and compared the soluble expression abilities of the different recombinant PnpC. The PnpC1 was purified by Ni-NTA affinity chromatography for the further biochemical charaterization. The results showed that the open reading frame of the pnpC1/2/3 contained 873 nucleotides coding for PnpC with identical molecular weight of 33 kDa. The three pnpC sequences had high homolog of 99%. At 37℃and 20℃, the recombinant pnpC1/2/3 could be highly expressed in E.coli BL21 with ortho ring cleavage activity towards catechol. At 37℃, the recombinant PnpC1/2/3 was predominantly expressed as inclusion bodies with little soluble expression. At 20℃, the recombinant PnpC1/3 was expressed mainly as soluble proteins, while the recombinant PnpC2 was still expressed as inclusion bodies. The amino acids sequence alignment indicated that the residue mutations between PnpC1 and C2 were T12I/A202V; PnpC2 and C3 were T12I/A202V/K230R; and PnpC1 and C3 was merely K230R. The activities of the recombinant PnpC1/2/3 towards catechol were 6.8,2.0 and 8.6 U/mL, respectively. The specific activities of them were 0.25,0.22 and 0.29 U/mg, respectively. The recombinant PnpC1 was purified to homogeneity by Ni-NTA affinity chromatography with specific activity 9.3 U/mg, purification fold 37.2, and yield rate 23.8%. The purified PnpC1 with ring cleavage activity to catechol exhibited optimum temperature of 45℃, optimum pH of 5.0, Km of 21.95μmol/L, and Vmax of 2.68μmol/(min-mg). The enzyme activity could be activated by Fe3+, Fe2+, Cu2+, Zn2+, Mn2+, and Co2+, while inhibited by Ni2+. This study provided a foundation for further investigating microbial mechanism of PNP degradation and for PnpC application of PNP bioremediation.